This invention relates to a high throughput screening platform and more particularly to a high throughput platform capable of in vivo genetic and chemical screens on specimen organisms such as zebrafish larvae and other teleosts.
Small animal models such as zebrafish (Danio Rerio) facilitate the study of complex processes on a large scale that cannot be replicated in vitro such as: organ development; neural degeneration and regeneration; stem cell proliferation and migration; cardiovascular, immune, endocrine, and nervous system functions; infectious disease progression; pathogenesis; cancer progression; and tissue specificity and toxicity of drugs. Several desirable attributes of zebrafish have fueled its popularity, including the animal's small size, optical transparency, aquatic habitat, and simplicity of culture. Zebrafish models of several human diseases have been developed1-11. Superscript numbers refer to the references included herewith. The contents of all these references are incorporated herein by reference. Lead compounds discovered by screening chemical compound libraries for efficacy in zebrafish disease models have been useful for pharmaceutical development because of the high level of conservation of drug activity between mammals and zebrafish12,13. The availability of large numbers of mutant strains and genetic manipulations such as gene overexpression, knockdown, and silencing make zebrafish a powerful model for genetic studies and for identification of the cellular targets of new compounds1,14,15. The significant advantages of zebrafish have fueled exponential growth of its use in experimental investigations over the last two decades1.
Several companies and academic labs are conducting genetic and compound screens on zebrafish larvae incubated in 96-well plates1,2,4,12. Because handling of zebrafish has been largely manual, typical high-content zebrafish screens are limited to only a few thousand compounds per week. Subcellular resolution assays require optical access to a specific region of the specimen for imaging or manipulation. Clear access is often impeded by intervening organs such as eyes and heart. Yolk and some organs exhibit significant autofluorescence. In addition, skin pigmentations can block the region of interest. Visualization of most of the regions requires orienting the zebrafish appropriately. However, current specimen orientation methods require embedding the sample in viscous media such as agar and/or manually rotating the fish with forceps. These processes are too slow and unreliable for high-throughput screens. In addition, specimens cannot be rapidly re-oriented once they are fixed, thus impeding visualization of organs from multiple angles. Examples of assays that require sample orientation and subcellular resolution imaging include in vivo monitoring of early tumor growth, neuronal degeneration, neurite regeneration, and stem cell proliferation and migration in tissues comprising the brain, eyes, heart, pancreas, kidneys, and liver.